1. Field of the Invention
This invention relates to method and apparatus for analyzing whole blood samples, and to methods and apparatus for evaluating constituents within a whole blood sample such as red blood cells, white blood cells, platelets, etc.
2. Description of the Prior Art
Physicians often utilize a blood test to determine the health of a patient. The complete Blood Count (CBC) is the most commonly performed clinical laboratory test in the United States and the world. Rapid identification and enumeration of the various components of biological fluids is an important diagnostic aim Minimal processing and handling of samples would contribute to the widespread use of such techniques.
Historically, a blood sample is taken from a patient and then sent to a laboratory for evaluation. Current CBC methods and instruments are highly evolved, using multi-channel, multi-detector flow system based technology. CBC instruments aspirate anticoagulated whole blood and divide it into several analysis streams to perform the different elements of the CBC. The elements include red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct) indices (MCV, MCH, MCHC), and red cell morphology; white blood cell count (WBC) and WBC differential count (enumeration of the different normal and abnormal white blood cell types; and platelet count.
The most common tests performed on blood samples taken from patients are the hematocrit (Hct), or the hemoglobin (Hb), which are often used interchangeably, depending upon the individual preference of the treating physician. They are used to determine anemia, to monitor conditions in which the blood loss occurs, chronic diseases, drug reactions, allergies, and the course of therapy.
The Hct of a sample of blood is defined as the ratio of the volume of erythrocytes (red blood cells) to that of the whole blood. It is expressed as a percentage or, preferably, as a decimal fraction. The units (L/L) are implied. The venous hematocrit agrees closely with the hematocrit obtained from a skin puncture; both are greater than the total body hematocrit.
The Hct and Hb are often provided along with the total red blood cell count (RBC) which is usually expressed in the form of a concentration—cells per unit volume of blood. Once these three values are known (Hct, Hb and RBC), three red blood cell indices are calculated. These indices are particularly useful in the morphologic characterization of anemias. These values include the mean cell volume (MCV) which is the average volume of red blood cells and is calculated from the Hct and the RBC. Utilizing the formula:MCV=Hct×1,000/RBC(in millions per μl)
The mean cell hemoglobin (MCH) may also be calculated and is the content of Hb in the average red blood cell; it is calculated from the Hb concentration and the RBC utilizing the following formula:
      M    ⁢                  ⁢    C    ⁢                  ⁢    H    =            Hb      ⁡              (                  in          ⁢                                          ⁢          g          ⁢                                          ⁢          per          ⁢                                          ⁢          liter                )                    R      ⁢                          ⁢      B      ⁢                          ⁢              C        ⁡                  (                      in            ⁢                                                  ⁢            millions            ⁢                                                  ⁢            per            ⁢                                                  ⁢            µl                    )                    
Another index calculable from the Hb and Hct is the mean cell hemoglobin concentration (MCHC). This index is the average concentration of Hb in a given volume of packed red blood cells. It is calculated using the following formula:
      M    ⁢                  ⁢    C    ⁢                  ⁢    H    ⁢                  ⁢    C    =            Hb      ⁡              (                  in          ⁢                                          ⁢                      g            /            dl                          )              Hct  
Other characteristics of red blood cells which are available utilizing today's testing methods include values for the variability of the MCV about a mean value and estimates of abnormality in red blood cell morphology.
The above described indices are discussed in much greater detail in John Bernard Henry, M.D., Clinical Diagnosis And Management By Laboratory Methods, Part IV (17th edition 1984).
Modern clinical laboratory instrumentation has been built to make these primary analyses simultaneously in vitro on blood samples removed from the patient and the calculated indices are readily produced by these instruments. The calculated indices are often the preferred data on which physicians base their conclusions about a patient's condition.
A large number of testing methods, instrumentation, and techniques have been used in measuring and approximating values for Hct, Hb and RBC. The most common method used to determine the Hct (the ratio of packed red blood cells to volume of whole blood) involves centrifugation wherein a given blood sample is placed into a centrifuge for five minutes at approximately 10,000 to 12,000 g. The volume is then calculated by measuring the level of the red blood cells as a ratio of the total volume.
Methods used in the art to determine the Hb in a sample of blood include the cyanmethemoglobin method, the oxyhemoglobin method and the method of measuring iron content of the sample. Of the above three methods, the first (the cyanmethoglobin method) is recommended by the International Committee for Standardization in Hematology. That method involves diluting a sample of blood in a solution of potassium ferricyanide and potassium cyanide. The potassium ferricyanide oxidizes hemoglobins and potassium cyanide provides cyanide ions to form hemiglobincyanide which has a broad absorption maximum at a wavelength of approximately 540 nm. The absorbance of the overall solution can then be measured in a photometer or spectrophotometer at 540 nm and compared with that of a standard hemoglobincyanide solution.
A large number of testing methods techniques and instruments have also been used in measurement of WBC counts, WBC differential counts and platelet counts. Rather than attempting to review the entire filed, refer to Henry (Ibid.)
Fully automated blood analysis systems are usually flow based and can cost more that $300,000. The systems require extensive calibration and control, maintenance, skilled operators and they have substantial costs associated with reagents, consumables and disposables. A large proportion of blood specimens processed by the systems requires further testing, depending of the laboratory policy regarding “flagging” criteria for certain findings and typically is from 10 to 50% of the samples. Retesting most frequently is required for direct visualization by a technologist, of abnormal RBC morphology or of the WBC differential due to an abnormal distribution of cell types or cells whose origin could be from hematologic or other malignancies or viral diseases. The additional testing includes retrieving the blood tube, removing blood from the tube and preparing and staining a smear on a glass slide, followed by visualization and analysis of the cells by a skilled technologist. The follow-up tests costs up to three times that of the initial instrumental analysis.
For follow-up, the sample is evaluated by smearing a small amount of blood on a slide, drying, fixing and staining it and then examination of the smear under a microscope. Unfortunately, the accuracy and reliability of the results depends largely on the technician's experience and technique. Additionally, blood smears are labor intensive and costly. Although the preparation and staining can be automated, examination remains a manual task.
A known reference method for evaluating a whole blood sample involves diluting a volume of whole blood, placing it within a “counting chamber”, and manually evaluating the constituent cells within the diluted sample. Dilution is necessary because the number and concentration of the red blood cells (RBCs) in whole blood vastly outnumber other constituent cells. To determine a WBC count, the whole blood sample must be diluted within a range of about one part blood to twenty parts diluent (1:20) up to a dilution of approximately 1:256, depending upon the exact technique used, and it is also generally necessary to selectively lyse the RBCs with one or more reagents. Lysing the RBCs effectively removes them from view so that the WBCs can be seen. To determine a platelet count, the blood sample must be diluted within a range of about 1:100 to approximately 1:50,000. Platelet counts do not, however, require a lysis of the RBCs in the sample. A disadvantage of this method of evaluating a whole blood sample is that the dilution process is time consuming and expensive. In addition, adding diluents to the whole blood sample increases the error probability within the sample data.
A modern method for evaluating a blood sample is impedance or optical flow cytometry. Flow cytometry involves circulating a diluted blood sample through one or more small diameter orifices, some with reagent addition streams flowing into them, each adjacent to an impedance type or an optical type sensor which evaluates the constituent cells as they pass through the orifice in single file. Different constituents may require different flow streams for their detection and estimation. For example, the blood sample must be diluted to mitigate the overwhelming number of the RBCs relative to the WBCs and the platelets for counting of each of these constituents. Further, separate streams of flow may be required for differentiation of white blood cells in order to do WBC differential counts. Each such stream imposes requirements for stream separation by valves or other means, pumps and detection devices. There are many variations of such processes in different flow based CBC instruments but all add complexity, numbers of moving parts, opportunities for component failure, needs for maintenance and costs. In addition, fluidics are required for different reagents to be added to different streams. Although more expedient and consistent than the above described reference methods, flow cytometry also possesses numerous disadvantages. Some of those disadvantages stem from the plumbing required to carry the sample to, and the fluid controls necessary to control the fluid flow rate through, the sensor means. The precise control of the sample flow is essential to the operation of the flow cytometer. The plumbing within flow cytometers may leak, potentially compromising the accuracy and the safety of the equipment. The fluid flow controls and dilution equipment, on the other hand, require periodic recalibration. The need for recalibration illustrates the potential for inaccurate results and the undesirable operating costs that exist with many presently available hematology analyzers which use flow cytometers. Another disadvantage is the volume of reagents required. Because of the large dilution ratios employed, correspondingly large volumes of liquid reagents are necessary. The large reagent volume increases the cost of the testing and creates a waste disposal problem.
Another approach to cellular analysis is volumetric capillary scanning as outlined in U.S. Pat. Nos. 5,547,849 and 5,585,246 for example, wherein a relatively undiluted sample of whole blood is placed into a capillary of known volume and thickness and is examined while the blood is in a quiescent state. This technique deals with the presence of the RBCs by limiting the scanning wavelengths to those with which the RBCs appear relatively transparent, and it requires that the sample be treated so that the RBCs do not aggregate during the measurement process. Thus, this technique is limited to the use of longer wavelength fluorescence, and there is no provision for the examination of RBCs and platelets or the examination of any cellular morphology.
U.S. Pat. No. 5,948,686 describes a method and apparatus for use in examining and obtaining information from a quiescent substantially undiluted anti-coagulated whole blood sample which is contained in a chamber having a top and bottom. Generally, the only reagents used were dyes, stains and anticoagulants, and these reagents were not added for the purpose of diluting the sample but rather were added to produce a reaction, an effect, or the like that facilitates the test at hand.
According to the invention, a method for evaluating constituents in undiluted anti-coagulated whole blood included the steps of: a) providing a sample chamber; b) admixing a sensible colorant with the sample of whole blood; c) inserting the admixed sample into the sample chamber; d) quiescently holding the admixed sample within the chamber until rouleaux and lacunae form within the sample; and e) evaluating a target constituent disposed within the lacunae.